Evidence that extracellular HSPB1 contributes to inflammation in alcohol-associated hepatitis.

Background and aims: Alcohol-associated hepatitis (AH) is the most life-threatening form of alcohol-associated liver disease (ALD). AH is characterized by severe inflammation attributed to increased levels of ethanol, microbes or microbial components, and damage-associated molecular pattern (DAMP) molecules in the liver. HSPB1 (Heat Shock Protein Family B (Small) Member 1; also known as Hsp25/27) is a DAMP that is rapidly increased in and released from cells experiencing stress, including hepatocytes. The goal of this study was to define the role of HSPB1 in AH pathophysiology. Methods: Serum HSPB1 was measured in a retrospective study of 184 heathy controls (HC), heavy alcohol consumers (HA), patients with alcohol-associated cirrhosis (AC), and patients with AH recruited from major hospital centers. HSPB1 was also retrospectively evaluated in liver tissue from 10 HC and AH patients and an existing liver RNA-seq dataset. Finally, HSPB1 was investigated in a murine Lieber-DeCarli diet model of early ALD as well as cellular models of ethanol stress in hepatocytes and hepatocyte-macrophage communication during ethanol stress. Results: Circulating HSPB1 was significantly increased in AH patients and levels positively correlated with disease-severity scores. Likewise, HSPB1 was increased in the liver of patients with severe AH and in the liver of ethanol-fed mice. In vitro , ethanol-stressed hepatocytes released HSPB1, which then triggered TNFα-mediated inflammation in macrophages. Anti-HSPB1 antibody prevented TNFα release from macrophages exposed to media conditioned by ethanol-stressed hepatocytes. Conclusions: Our findings support investigation of HSPB1 as both a biomarker and therapeutic target in ALD. Furthermore, this work demonstrates that anti-HSPB1 antibody is a rational approach to targeting HSPB1 with the potential to block inflammation and protect hepatocytes, without inactivating host defense. HIGHLIGHTS: HSPB1 is significantly increased in serum and liver of patients with alcohol-associated hepatitis.Ethanol consumption leads to early increases in HSPB1 in the mouse liver.Hepatocytes subjected to ethanol stress release HSPB1 into the extracellular environment where it activates TNFα-mediated inflammation in macrophages.Anti-HSPB1 antibody blocks hepatocyte-triggered TNFα in a model of hepatocyte-macrophage communication during ethanol stress.

Genome Restructuring around Innate Immune Genes in Monocytes in Alcohol-associated Hepatitis.

Many inflammatory genes in the immune system are clustered in the genome. The 3D genome architecture of these clustered genes likely plays a critical role in their regulation and alterations to this structure may contribute to diseases where inflammation is poorly controlled. Alcohol-associated hepatitis (AH) is a severe inflammatory disease that contributes significantly to morbidity in alcohol associated liver disease. Monocytes in AH are hyper-responsive to inflammatory stimuli and contribute significantly to inflammation. We performed high throughput chromatin conformation capture (Hi-C) technology on monocytes isolated from 4 AH patients and 4 healthy controls to better understand how genome structure is altered in AH. Most chromosomes from AH and healthy controls were significantly dissimilar from each other. Comparing AH to HC, many regions of the genome contained significant changes in contact frequency. While there were alterations throughout the genome, there were a number of hotspots containing a higher density of changes in structure. A few of these hotspots contained genes involved in innate immunity including the NK-gene receptor complex and the CXC-chemokines. Finally, we compare these results to scRNA-seq data from patients with AH challenged with LPS to predict how chromatin conformation impacts transcription of clustered immune genes. Together, these results reveal changes in the chromatin structure of monocytes from AH patients that perturb expression of highly clustered proinflammatory genes.

Proteomics identifies complement protein signatures in patients with alcohol-associated hepatitis.

Diagnostic challenges continue to impede development of effective therapies for successful management of alcohol-associated hepatitis (AH), creating an unmet need to identify noninvasive biomarkers for AH. In murine models, complement contributes to ethanol-induced liver injury. Therefore, we hypothesized that complement proteins could be rational diagnostic/prognostic biomarkers in AH. Here, we performed a comparative analysis of data derived from human hepatic and serum proteome to identify and characterize complement protein signatures in severe AH (sAH). The quantity of multiple complement proteins was perturbed in liver and serum proteome of patients with sAH. Multiple complement proteins differentiated patients with sAH from those with alcohol cirrhosis (AC) or alcohol use disorder (AUD) and healthy controls (HCs). Serum collectin 11 and C1q binding protein were strongly associated with sAH and exhibited good discriminatory performance among patients with sAH, AC, or AUD and HCs. Furthermore, complement component receptor 1-like protein was negatively associated with pro-inflammatory cytokines. Additionally, lower serum MBL associated serine protease 1 and coagulation factor II independently predicted 90-day mortality. In summary, meta-analysis of proteomic profiles from liver and circulation revealed complement protein signatures of sAH, highlighting a complex perturbation of complement and identifying potential diagnostic and prognostic biomarkers for patients with sAH.

Membrane-bound O-acyltransferase 7 (MBOAT7) shapes lysosomal lipid homeostasis and function to control alcohol-associated liver injury.

Recent genome-wide association studies (GWAS) have identified a link between single-nucleotide polymorphisms (SNPs) near the MBOAT7 gene and advanced liver diseases. Specifically, the common MBOAT7 variant (rs641738) associated with reduced MBOAT7 expression is implicated in non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis. However, the precise mechanism underlying MBOAT7-driven liver disease progression remains elusive. Previously, we identified MBOAT7-driven acylation of lysophosphatidylinositol lipids as key mechanism suppressing the progression of NAFLD (Gwag et al., 2019). Here, we show that MBOAT7 loss of function promotes ALD via reorganization of lysosomal lipid homeostasis. Circulating levels of MBOAT7 metabolic products are significantly reduced in heavy drinkers compared to healthy controls. Hepatocyte- (Mboat7-HSKO), but not myeloid-specific (Mboat7-MSKO), deletion of Mboat7 exacerbates ethanol-induced liver injury. Lipidomic profiling reveals a reorganization of the hepatic lipidome in Mboat7-HSKO mice, characterized by increased endosomal/lysosomal lipids. Ethanol-exposed Mboat7-HSKO mice exhibit dysregulated autophagic flux and lysosomal biogenesis, associated with impaired transcription factor EB-mediated lysosomal biogenesis and autophagosome accumulation. This study provides mechanistic insights into how MBOAT7 influences ALD progression through dysregulation of lysosomal biogenesis and autophagic flux, highlighting hepatocyte-specific MBOAT7 loss as a key driver of ethanol-induced liver injury.

Infective Endocarditis in Patients Addicted to Injected Opioid Drugs.

BACKGROUND: Persons who inject drugs and require surgery for infective endocarditis have 2 potentially lethal diseases. Current postoperative rehabilitation efforts seem ineffective in preventing loss to follow-up, injection drug use relapse (relapse), and death. OBJECTIVES: The purpose of this study was to characterize drug use, psychosocial issues, surgical outcome, and postoperative addiction management, as well as loss to follow-up, relapse, and mortality and their risk factors. METHODS: From January 2010 to June 2020, 227 persons who inject drugs, age 36 ± 9.9 years, underwent surgery for infective endocarditis at a quaternary hospital having special interest in developing addiction management programs. Postsurgery loss to follow-up, relapse, and death were assessed as competing risks and risk factors identified parametrically and by machine learning. CIs are 68% (±1 SE). RESULTS: Heroin was the most self-reported drug injected (n = 183 [81%]). Psychosocial issues included homelessness (n = 56 [25%]), justice system involvement (n = 150 [66%]), depression (n = 118 [52%]), anxiety (n = 104 [46%]), and post-traumatic stress disorder (n = 33 [15%]). Four (1.8%) died in-hospital. Medication for opioid use disorder prescribed at discharge increased from 0% in 2010 to 100% in 2020. At 1 and 5 years, conditional probabilities of loss to follow-up were 16% (68% CI: 13%-22%) and 59% (68% CI: 44%-65%), relapse 32% (68% CI: 28%-34%) and 79% (68% CI: 74%-83%), and mortality 21% (68% CI: 18%-23%) and 68% (68% CI: 62%-72%). Younger age, heroin use, and lower education level were predictors of relapse. CONCLUSIONS: Infective endocarditis surgery can be performed with low mortality in persons who inject drugs, but addiction is far more lethal. Risk of loss to follow-up and relapse require more effective addiction strategies without which this major loss to society will continue.

Membrane Bound O-Acyltransferase 7 (MBOAT7) Shapes Lysosomal Lipid Homeostasis and Function to Control Alcohol-Associated Liver Injury.

Several recent genome-wide association studies (GWAS) have identified single nucleotide polymorphism (SNPs) near the gene encoding membrane-bound O -acyltransferase 7 ( MBOAT7 ) that is associated with advanced liver diseases. In fact, a common MBOAT7 variant (rs641738), which is associated with reduced MBOAT7 expression, confers increased susceptibility to non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis in those chronically infected with hepatitis viruses B and C. The MBOAT7 gene encodes a lysophosphatidylinositol (LPI) acyltransferase enzyme that produces the most abundant form of phosphatidylinositol 38:4 (PI 18:0/20:4). Although these recent genetic studies clearly implicate MBOAT7 function in liver disease progression, the mechanism(s) by which MBOAT7-driven LPI acylation regulates liver disease is currently unknown. Previously we showed that antisense oligonucleotide (ASO)-mediated knockdown of Mboat7 promoted non-alcoholic fatty liver disease (NAFLD) in mice (Helsley et al., 2019). Here, we provide mechanistic insights into how MBOAT7 loss of function promotes alcohol-associated liver disease (ALD). In agreement with GWAS studies, we find that circulating levels of metabolic product of MBOAT7 (PI 38:4) are significantly reduced in heavy drinkers compared to age-matched healthy controls. Hepatocyte specific genetic deletion ( Mboat7 HSKO ), but not myeloid-specific deletion ( Mboat7 MSKO ), of Mboat7 in mice results in enhanced ethanol-induced hepatic steatosis and high concentrations of plasma alanine aminotransferase (ALT). Given MBOAT7 is a lipid metabolic enzyme, we performed comprehensive lipidomic profiling of the liver and identified a striking reorganization of the hepatic lipidome upon ethanol feeding in Mboat7 HSKO mice. Specifically, we observed large increases in the levels of endosomal/lysosomal lipids including bis(monoacylglycero)phosphates (BMP) and phosphatidylglycerols (PGs) in ethanol-exposed Mboat7 HSKO mice. In parallel, ethanol-fed Mboat7 HSKO mice exhibited marked dysregulation of autophagic flux and lysosomal biogenesis when exposed to ethanol. This was associated with impaired transcription factor EB (TFEB)-mediated lysosomal biogenesis and accumulation of autophagosomes. Collectively, this works provides new molecular insights into how genetic variation in MBOAT7 impacts ALD progression in humans and mice. This work is the first to causally link MBOAT7 loss of function in hepatocytes, but not myeloid cells, to ethanol-induced liver injury via dysregulation of lysosomal biogenesis and autophagic flux.

Dysregulated meta-organismal metabolism of aromatic amino acids in alcohol-associated liver disease.

BACKGROUND: Chronic alcohol consumption impairs gut barrier function and perturbs the gut microbiome. Although shifts in bacterial communities in patients with alcohol-associated liver disease (ALD) have been characterized, less is known about the interactions between host metabolism and circulating microbe-derived metabolites during the progression of ALD. METHODS: A large panel of gut microbiome-derived metabolites of aromatic amino acids was quantified by stable isotope dilution liquid chromatography with online tandem mass spectrometry in plasma from healthy controls (n = 29), heavy drinkers (n = 10), patients with moderate (n = 16) or severe alcohol-associated hepatitis (n = 40), and alcohol-associated cirrhosis (n = 10). RESULTS: The tryptophan metabolites, serotonin and indole-3-propionic acid, and tyrosine metabolites, p-cresol sulfate, and p-cresol glucuronide, were decreased in patients with ALD. Patients with severe alcohol-associated hepatitis and alcohol-associated cirrhosis had the largest decrease in concentrations of tryptophan and tyrosine-derived metabolites compared to healthy control. Western blot analysis and interrogation of bulk RNA sequencing data from patients with various liver pathologies revealed perturbations in hepatic expression of phase II metabolism enzymes involved in sulfonation and glucuronidation in patients with severe forms of ALD. CONCLUSIONS: We identified several metabolites decreased in ALD and disruptions of hepatic phase II metabolism. These results indicate that patients with more advanced stages of ALD, including severe alcohol-associated hepatitis and alcohol-associated cirrhosis, had complex perturbations in metabolite concentrations that likely reflect both changes in the composition of the gut microbiome community and the ability of the host to enzymatically modify the gut-derived metabolites.

Altered anti-viral immune responses in monocytes in overweight heavy drinkers.

Alcohol abuse causes increased susceptibility to respiratory syndromes like bacterial pneumonia and viral infections like SARS-CoV-2. Heavy drinkers (HD) are at higher risk of severe COVID-19 if they are also overweight, yet the molecular mechanisms are unexplored. Single-cell RNA-sequencing (scRNA-seq) was performed on peripheral blood mononuclear cells from lean or overweight HD and healthy controls (HC) after challenge with a dsRNA homopolymer (PolyI:C) to mimic a viral infection and/or with lipopolysaccharide (LPS). All monocyte populations responded to both PolyI:C and LPS with pro-inflammatory gene expression. However, the expression of interferon-stimulated genes, essential for inhibiting viral pathogenesis, was greatly reduced in overweight patients. Interestingly, the number of upregulated genes in response to the PolyI:C challenge was far greater in monocytes from HD compared to HC, including much stronger pro-inflammatory cytokine and interferon-γ signaling responses. These results suggest that increased body weight reduced anti-viral responses while heavy drinking increased pro-inflammatory cytokines.

Peripheral blood mononuclear cell mitochondrial dysfunction in acute alcohol-associated hepatitis.

BACKGROUND: Patients with acute alcohol-associated hepatitis (AH) have immune dysfunction. Mitochondrial function is critical for immune cell responses and regulates senescence. Clinical translational studies using complementary bioinformatics-experimental validation of mitochondrial responses were performed in peripheral blood mononuclear cells (PBMC) from patients with AH, healthy controls (HC), and heavy drinkers without evidence of liver disease (HD). METHODS: Feature extraction for differentially expressed genes (DEG) in mitochondrial components and telomere regulatory pathways from single-cell RNAseq (scRNAseq) and integrated 'pseudobulk' transcriptomics from PBMC from AH and HC (n = 4 each) were performed. After optimising isolation and processing protocols for functional studies in PBMC, mitochondrial oxidative responses to substrates, uncoupler, and inhibitors were quantified in independent discovery (AH n = 12; HD n = 6; HC n = 12) and validation cohorts (AH n = 10; HC n = 7). Intermediary metabolites (gas-chromatography/mass-spectrometry) and telomere length (real-time PCR) were quantified in subsets of subjects (PBMC/plasma AH n = 69/59; HD n = 8/8; HC n = 14/27 for metabolites; HC n = 13; HD n = 8; AH n = 72 for telomere length). RESULTS: Mitochondrial, intermediary metabolite, and senescence-regulatory genes were differentially expressed in PBMC from AH and HC in a cell type-specific manner at baseline and with lipopolysaccharide (LPS). Fresh PBMC isolated using the cell preparation tube generated optimum mitochondrial responses. Intact cell and maximal respiration were lower (p ≤ .05) in AH than HC/HD in the discovery and validation cohorts. In permeabilised PBMC, maximum respiration, complex I and II function were lower in AH than HC. Most tricarboxylic acid (TCA) cycle intermediates in plasma were higher while those in PBMC were lower in patients with AH than those from HC. Lower telomere length, a measure of cellular senescence, was associated with higher mortality in AH. CONCLUSION: Patients with AH have lower mitochondrial oxidative function, higher plasma TCA cycle intermediates, with telomere shortening in nonsurvivors.

Reported firearm access among patients admitted to a dual diagnosis medically-assisted withdrawal unit over five years.

Background: Up to one-third of firearm-related suicides were carried out by individuals who had consumed alcohol shortly before their death. Despite the critical role of firearm access screening in suicide risk assessment, few studies have examined firearm access among patients with substance use disorders. This study examines the rates of firearm access among those admitted to a co-occurring diagnosis unit over a five year period. Methods: All patients admitted to a co-occurring disorders inpatient unit from 2014 to mid-2020 were included. An analysis contrasting the differences among patients reporting firearms was performed. A multivariable logistic regression model using factors from initial admission were chosen based on clinical relevance, past firearms research, and statistical significance on bivariate analysis was used. Results: Over the study period there were 7332 admissions representing 4055 patients. Documentation of firearm access was completed in 83.6% of admissions. Firearm access was reported in 9.4% of admissions. Patients reporting firearm access were more likely to report never having suicidal ideation (p = 0.001), be married (p = <0.001), and report no past history of suicide attempts (p = <0.001). The full logistic regression model revealed that being married (OR: 2.29 and p < 0.0001) and employed (OR: 1.51 and p = 0.024) were factors associated with firearms access. Conclusions: This is one of the largest reports assessing factors associated with firearm access among those admitted to a co-occurring disorders unit. Firearm access rates in this population appear lower than rates in the general population. The roles employment and marital status play in firearm access deserve future attention.

Diminished function of cytotoxic T- and NK- cells in severe alcohol-associated hepatitis.

Aim: Metabolic liver diseases, including alcohol- and non-alcoholic fatty liver diseases (ALD/NAFLD), are characterized by inflammation and decreased ability to prevent infections. Patients with severe alcohol-associated hepatitis (sAH) are particularly susceptible to infections while undergoing treatment with steroids. Understanding the immunological mechanisms for these responses is critical to managing the treatment of patients with metabolic liver diseases. Cytotoxic NK cells and CD8 T cells, using cytolytic granules, serve an important immunological role by killing infected cells, including monocytes. However, patients with sAH have dysfunctional NK cells, which cannot kill target cells, though the mechanism is unknown. Method: We performed an exploratory study using single-cell RNA-seq (scRNA-seq) (n = 4) and multi-panel intracellular flow cytometry (n = 7-8 for all patient groups) on PBMCs isolated from patients with sAH and healthy controls (HC). Results: ScRNA-seq revealed receptors in NK cells and CD8 T cells required for cytotoxic cell recognition of activated monocytes were downregulated in patients with sAH compared to healthy controls. Granulysin was the most downregulated gene in both NK cells and effector CD8 T cells. In NK cells from HC, expression of granulysin, perforin, and granzymes A and B was highly correlated; however, in sAH, these genes lost coordinate expression, indicative of dysfunctional cytolytic granule formation. Finally, the expression of cytolytic granule proteins in NK cells was decreased from sAH, indicating reduced cytolytic granules. Conclusion: Together, these results suggest a loss of cytotoxic cell function in PBMCs from sAH that may contribute to a decreased ability to communicate with other immune cells, such as monocytes, and prevent the killing of infected cells, thus increasing the risk of infection.

E-cigarettes: Safe to recommend to patients?

Electronic cigarettes (e-cigarettes)--devices that generate a nicotine vapor that can be inhaled by the user in a fashion that mimics the experience of smoking--are increasing in popularity, and many people seem to view them as reasonable alternatives to nicotine replacement therapy to help them refrain from smoking. Physicians should not encourage such a view. E-cigarettes are unregulated nicotine delivery systems that have never been subjected to any kind of testing of safety or of efficacy as nicotine replacement therapy. Moreover, for young people who have never smoked, these devices could potentially serve as a gateway drug.

Synthetic legal intoxicating drugs: the emerging 'incense' and 'bath salt' phenomenon.

Synthetic legal intoxicating drugs (SLIDs), such as those commonly contained in products sold over the counter as "bath salts" and "incense," have risen tremendously in popularity in the past few years. These drugs can have powerful adverse effects, including acute psychosis with delusions, hallucinations, and potentially dangerous, bizarre behavior.

Sleep disorders in substance abusers: how common are they?

UNLABELLED: Substance abuse is a major public health problem with high morbidity and mortality. Comorbid disorders are suspected to cause a high relapse rate. Subjects with sleep disorders tend to self medicate with alcohol and tranquilizers to promote sleep or abuse stimulants to stay awake during the day. Substance abuse can, in turn, cause sleep disturbances, which can result in relapse. No studies have systematically studied the prevalence of various sleep disorders in these subjects. METHODS: This is a cross-sectional study conducted at the Alcohol and Drug Recovery Center (ADRC) at Cleveland Clinic, Cleveland, Ohio. Subjects with active substance abuse and the ability to consent were recruited to complete a comprehensive sleep disorder questionnaire, including a general medical, psychiatric, and substance abuse history as well as validated scales (e.g., Insomnia Severity Index, Pittsburgh Sleep Quality Index (PSQI), Berlin Questionnaire for sleep apnea and restless legs). RESULTS: Thirty patients completed the survey so far. The most commonly abused substance was alcohol (80%) followed by narcotics (40%); about 66 percent were polysubstance users. Forty-six percent of the patients reported using substance to self-medicate sleep problems. The prevalence of various sleep disorders in this population along with the prevalence in general population in parenthesis are as follows: Sleep impairment (PSQI>5) was noted in 96 percent (15%) of the subjects, and 56 percent (10-15%) had insomnia of moderate-to-severe degree. Symptoms suggestive of sleep apnea were reported in 53 percent (4-6%) of the subjects and restless leg syndrome symptoms in 33 percent (10%). CONCLUSION: Substance abuse is on the rise and affects every aspect of society. Our study has, for the first time, systematically evaluated various sleep disorders in these subjects who seem 5 to 10 times more likely to have sleep disorders. Diagnosing and treating sleep disorders will have a huge impact in inducing remission. However, this study has significant limitations, including a small number of subjects, subjective data collected via questionnaires, and no long-term follow up, which makes it difficult to draw conclusions.